Fluorescence resonance energy transfer (abbreviated FRET), also known as Förster resonance energy transfer (named after its discoverer Theodor Forster) is a mechanism describing the transfer of excitation energy from one molecule to another without the need for fluorescence and re-absorption. According to Förster, energy transfer proceeds via dipole-dipole coupling of the donor fluorescence dipoles with the acceptor absorption dipoles. Thus, the phenomenon of FRET is always a non-radiative energy transfer. A donor chromophore, initially in its electronically excited state after having absorbed light of a certain wavelength may transfer energy radiationless to an acceptor, whereupon the acceptor is promoted to its electronically excited state. Subsequently, the electronically excited state of the acceptor decays so that in turn energy is emitted. The efficiency of FRET depends on many parameters which can be grouped as follows: the distance between the donor and the acceptor; the spectral overlap of the donor emission spectrum and the acceptor absorption spectrum; and the relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment.
In conventional FRET technology donor and acceptor are both fluorophores. Accordingly, energy absorbed by a donor fluorophore as light of a certain wavelength (absorption wavelength) is transferred to the acceptor. By absorption of the transferred energy the acceptor is promoted to an electronically excited state which subsequently decays whereupon the energy transferred to the acceptor is emitted as light of a particular wavelength (emission wavelength). The emission wavelength is shifted to longer wavelength in comparison to the absorption wavelength. When donor and acceptor are in close proximity (e.g., 1-10 nm) due to the interaction of the chromophores, the acceptor emission is predominantly observed because of the FRET from the donor to the acceptor. Accordingly, the phenomenon of FRET can be detected via a decrease of donor fluorescence or an increase of acceptor fluorescence.
In a specific form of FRET a so-called quencher is applied instead of a fluorescent acceptor (J. R. Lakowicz, Principles of Fluorescence, 2nd edition, Kluwer Academic Plenum Publishers, New York, 1999). A quencher is a molecule which absorbs the energy transferred from the donor (also called reporter) but instead of in turn emitting light it quenches fluorescence. Accordingly, in a reporter-quencher system the donor transfers energy to the quencher. Thereby, the donor returns to the ground state and generates the excited state of the quencher. Subsequently, the excited state of the quencher decays non-radiatively (dark quencher). In non-radiative or dark decay, energy is given off via molecular vibrations (heat). Since the concentration of quenchers in a probe is typically in the range of μM or less, the heat of radiationless decay is too small to affect the temperature of the solution. According to the Förster equation such a fluorescence quenching also depends on the distance between donor and acceptor. In contrast to the FRET technique mentioned above not the emission of the acceptor but only the one of the donor is measured: the more the chromophores move apart from each other, the weaker the energy transfer gets so that the fluorescence of the donor correspondingly increases.
Until the last few years, quenchers have typically been fluorescent dyes, for example, fluorescein as the reporter and rhodamine as the quencher (FAM/TAMRA probes). One of the best known quenchers is TAMRA (tetramethyl-rhodamine) which is used to lower the emission of the reporter dye. Due to its properties TAMRA is suitable as quencher for FAM (carboxyfluorescein), HEX (hexachlorofluorescein), TET (tetrachloro-fluorescein), JOE (5′-Dichloro-dimethoxy-fluorescein) and Cy3-dyes (cyanine).
The usefulness of TAMRA is, however, limited because of its broad emission spectrum which reduces its capabilities in multiplexing (when two or more reporter-quencher probes are used together). Its intrinsic fluorescence contributes to the background signal which leads to decreased signal dynamics and thus, potentially reduces the sensitivity of assays based on TAMRA.
Dark quenchers offer a solution to this problem because they do not occupy an emission bandwidth. Furthermore, dark quenchers enable multiplexing. A typical dark quencher is DABCYL (4-[[4-(dimethylamino)-phenyl]-azo]-benzoic acid) which is often used in combination with molecular beacons. DABCYL quenches dyes in a range of from 380 to 530 nm. Accordingly, even fluorophors having longer wave length emission such as Cy3-dyes can be better quenched by DABCYL. However, DABCYL has an inadequate absorption band that overlaps very poorly with fluorophores emitting above 480 nm. A further non-fluorescent dye is Eclipse Quencher (4-[[2-chloro-4-nitro-phenyl]-azo]-aniline (Epoch Biosciences, Inc.) which has an absorption maximum at 530 nm and efficiently quenches over a spectrum from 520 to 670 nm.
An improvement over the dark quenchers mentioned above are the Black Hole Quenchers, such as BHQ-1 ([(4-(2-nitro-4-methyl-phenyl)-azo)-yl-((2-methoxy-5-methyl-phenyl)-azo)]-aniline) and BHQ-2 ([(4-(1-nitro-phenyl)-azo)-yl-((2,5-dimethoxy-phenyl)-azo)]-aniline) (all available from Biosearch Technologies, Inc.) which are capable of quenching across the entire visible spectrum. These non-fluorescent acceptors are often applied as alternative to fluorescent acceptors in order to decrease background fluorescence and in this way sensitivity.
The disadvantage of the known non-fluorescent quenchers is, however, their insufficient quenching behavior resulting in high background which in turn leads to limited signal dynamics.
Therefore, one object of the present invention was the provision of new quenchers, preferably with a low background signal and/or high quenching efficiency. Additionally, in a preferred embodiment they may be coupled to biomolecules or a solid support for FRET.